Cocoa beans (Theobroma Cacao L) are one of the most important cash crops around the world. The process for commercial consumption of cocoa begin with the most important postharvest operation of fermentation where the ripened cocoa beans with their surrounding pulp are removed from the pods and placed in piles or bins, allowing access to microorganisms for fermentation. The duration of fermentation may last even seven days to generate unique flavor precursors which eventually provide familiar chocolate taste upon drying. Drying is conventionally performed by spreading the beans out in the sun from five to seven days. The dried beans are cleaned, roasted, and graded. Subsequently the shell of each bean is removed by the process called winnowing to extract the nib. The nibs are ground and liquefied, resulting in pure chocolate in fluid form, referred to as chocolate liquor. The liquor can be further pressed into two components, viz. cocoa solids or pressed cake and cocoa butter. The pressed cake is again processed for adjusting the pH (if required) and powdered to cocoa powder, which is normally the cocoa ingredient for the most majority of cocoa based food products. A large number of patents have been published on specific processes by which fermented cocoa beans can be converted to cocoa powder with unique taste and aroma.
Cocoa beans are very nutritious and contain chockfull of antioxidants, dietary fiber and proteins. On an average, cocoa beans contains 50 to 60% fat, 14 to 16% proteins, 16 to 20% dietary fiber, 5 to 8% carbohydrates and phytochemicals such as polyphenols (4 to 10%), theobromine (1 to 2%), caffeine (0.5 to 1%) etc. Upon fermentation, the carbohydrate profile and polyphenol load decreases. For instance, the polyphenol content of fermented cocoa is less than 3%, as compared to unfermented dried cocoa beans (7 to 9%). Further processes for the production of cocoa powder such as drying, roasting, grinding, alkalization etc are proved to reduce the polyphenol load to less than 1%. Thus, it has been estimated that, more than 80% of the antioxidant phytonutrients, mainly polyphenols belonging to the flavanoid family, especially the flavan-3-ol monomers, (−)-epicatechin and (+)-catechin and several procyanidin oligomers built upon these monomeric units are getting destroyed during the process of cocoa powder manufacture (Kim and Keeney. 1984; Hansen et. al. 1998). Moreover, some considerable fraction of flavonoids are converted to insoluble forms by the action of polyphenol oxidase, which are very poorly bioavailable upon consumption.
The preservation or enhancement of cocoa flavonoids is of great importance since these compounds are found to offer many health beneficial effects. Cocoa polyphenols have been reported to be good antioxidant with greater activity than tea and red wine polyphenols (Lee et al 2003). They are reported as general cardioprotective with wide range of biological activities, including eicosanoid synthesis modulation, increasing nitric oxide synthesis, lowering low-density lipoprotein oxidation, inhibiting platelet activation, stimulation of the production of anti-inflammatory cytokines, and inhibition of the production of certain proinflammatory cytokines etc. Its positive effects in various other disease states such as hypertension, brain function, blood sugar management, body weight management etc have also been substantiated. The skin benefits includes general improvements in skin health such as increased firmness, increased elasticity, reduced wrinkles (including wrinkle width and/or volume), reduced fine lines, increased hydration, decreased skin roughness, decreased scaling, improved skin smoothness, improved skin structure etc. (Waterhouse et al., 1996; Kondo et al., 1996; Mao et al., 1999; Karim et al., 2000; Mao et al., 2000; Rein et al., 2000; Schramm et al., 2001; Wan et al., 2001).
Various solvent systems have been recommended for extraction of polyphenols. Zieglader et al. used methanol to extract the polyphenols from cocoa beans at ambient temperature. The extract was found to contain monomer tannin precursors (catechins, anthocyanidins and their soluble condensates), and used as an additive for oil to preserve from oxidation (See “Antioxidative effects of cocoa”, Rev Choc Confect Bak, 8: 3-6, 1983). Griffiths et al. used methanol extracts of ripe cocoa nibs, again obtained at ambient temperatures, for characterization of polyphenols in cocoa and other plants (Biochemical J. 74: 362-365, 1960). Rigaud et al. made extracts from lyophilized cocoa beans and grape seeds, and noted that the use of methanol as a solvent precluded the presence of the higher oligomers (J. Chromatography 654:255-60, 1993). Jalal & Collin prepared extracts from different parts of the cocoa plant in order to analyze the polyphenols present in each part of the plant. The extraction was carried out using 70% cold methanol, followed by ethyl acetate (Phytochemistry, 16:1377-1380, 1977). Acetone/water has also been used for cocoa bean extractions. Clapperton et al. reported the extraction of defatted cocoa powder made from fermented cocoa beans using cold 70% v/v acetone/water (Polyphenols and Cocoa Flavor, Groupe Polyphenols, XVI. sup Intern. Conf., Lisbon, Portugal, Jul. 13-16, 1992).
Many methods employing above solvents and their optimized ratios and extraction conditions such as temperature, pressure etc have also been patented for the isolation of polyphenols from cocoa beans. Below listed prior arts describe various processes for the isolation of polyphenols, mainly flavonoids consisting flavan-3-ols and procyanidin oligomers from cocoa beans and their uses. The prior arts in concentrating polyphenols generally involves harvest of ripened cocoa fruit, special treatment to inhibit polyphenoloxidase enzyme, drying without fermentation and subsequent solvent extraction under controlled conditions to obtain extracts of original composition endowed with very useful properties.                1. 2008/0193629 A1 reports a water/alcohol or water/acetone extraction of defatted unfermented cocoa beans and further purification of resulting aqueous extracts using microfiltration with molecular weight cut off membranes followed by ultrafiltration and nano filtration to enrich polyphenols to 80% level. The patent is specific for the preparation of polyphenol enriched extracts from unfermented cocoa beans containing higher polyphenol content. It does not say anything about the recovery and process from fermented cocoa beans containing relatively low amount of polyphenol content. The process include separate defatting step using solvents like hexane and also generate more than 90% of the defatted cocoa material as a waste. The process of the invention also requires expensive plant/equipment's installations for the production.        2. U.S. Pat. No. 6,627,232 B1, describes extraction of defatted, unroasted, unfermented cocoa beans with organic solvents such as hexane, acetone, methanol, ethyl acetate etc and their aqueous combinations at various percentage. Extraction solvent systems and conditions such as temperature and pH were optimized to obtain lower molecular weight procyanidin enriched extracts and higher molecular weight procyanidin rich extracts separately. The patented invention has used counter-current batch extraction process. This patent is also specific for the preparation of procyanidin oligomers enriched extracts from unfermented cocoa beans containing higher polyphenol content and does not say anything about the recovery and process from fermented cocoa beans containing relatively low amount of polyphenol content. The process include separate defatting step using solvents like hexane and also generate more than 90% of the defatted cocoa material as a waste. Though the invention has detailed several solvent systems for the extraction of procyanidin oligomers, it does not say anything about the possibility of use of techniques such as ultrasonication or enzymes for the quantitative extraction of useful phytochemicals such as polyphenols and methylxanthines. The invention does not say anything about the process conditions or steps to be followed to make use of the spent of cocoa beans after extracting the procyanidins and methylxanthines to useful ingredients such as dietary fiber, proteins etc. The advantage of the present invention is that it is a green process of ultrasound mediated direct extraction of cocoa beans for highly efficient extraction, purification and formulation of cocoa phytonutrients and the optimized process for the recovery of useful ingredients like dietary fiber, proteins etc, leaving absolutely no waste.        3. U.S. Pat. No. 7,368,144 B2/2008 describes a method for obtaining cocoa bean polyphenol extracts from fresh beans and resulting extracts and uses thereof. It suggests various solvent systems of extraction and compositions comprising polyphenols, xanthins and lipids. The method does not provide any analysis or the characterization of the product in terms of procyanidines and the purity of polyphenol. The patent only describes a method of making a crude extract such as oleoresins and its analysis with respect to fatty acid, phytosterol, polyphenol and xanthines and some cosmetic effect of the said extracts. It does not give any information or analysis of total polyphenols, procyanidines and the recovery of the active ingredient in the process. The patent also does not provide any procedure for enhancing the purity of the phytonutrients such as polyphenols or methylxanthines. Since polyphenols are proved to be an important group of active principles in cocoa beans, higher purity is useful for reducing the dosage and enhancing the activity and also for various formulations with enhanced solubility, stability and bioavailability, for functional food or dietary supplement applications. Moreover, the patent does not provide conditions to make use of the spent of the polyphenol extract as value added fractions of dietary fiber, proteins etc.        4. US2008/0021227 A1 describes the isolation of polyphenols containing procyanidines and their use as antineoplastic agents in cancer treatment. This is a lab scale isolation method in which chlorinated solvents like chloroform are used. In addition, extensive separation of procyanidines using reverse phase and normal phase chromatography and sephadex separation are used. The process does not have any commercial validity as it is a small scale process.        5. US 2007/0134400 A1 details about the fractionation of cocoa beans into various fractions such as butter, cocoa solids, cocoa liquor etc and the polyphenol analysis of each fraction. A number of food recipes for polyphenol incorporation were also suggested. But, the procedure given is only for the selective extraction of procyanidines from specially processed unfermented cocoa beans and does not say anything about the possibility of extraction from normal fermented cocoa beans used for majority of the cocoa based applications or to make cocoa powder. Only small batch scale extraction procedure was given and does not speak about either the possibility of continuous extraction, or the use of techniques like ultrasonication to improve the speed, yield and purity of the extracts. The patent does not mention about the other useful components in cocoa beans such as methylxanthines, dietary fiber or proteins.        6. US 2008/0051587 A1 is basically an existing method with enhanced results (a counter current organic solvent extraction) for the selective extraction of procyanidines. Parameters like temperature, pH, solvent etc are optimized for monomer and oligomer enriched extracts. ie, selective extraction of oligomers according to size have been reported and were analysed by HPLC. However, no data regarding the yield or the purity level of procyanidines have been given. Theobromine levels in the extract were also not mentioned.        7. US 2008/0193629A1 is a patent for a method that details a blanching step of cocoa beans to reduce polyphenol oxidase activity, followed by defatting and extraction with aqueous alcohol or acetone. The extract was further subjected to ultrafiltration, nanofiltration and microfiltration to get the polyphenol enriched extract. The patent suggested the use of supercritical carbondioxide extraction, hydraulic press and solvent extraction to remove fat or butter. The defatted fractions are extracted and subjected to membrane filtration to get the polyphenol enriched extract. Composition of the final product with respect to polyphenol, procyanidines, and theobromine are not mentioned here also.        8. US 2004/096566 is a process for carrying out the extraction under specific conditions that makes it possible to process cocoa beans to provide products with a high polyphenol content enriched (in comparison to the initial content of the beans) with certain useful lipid derivatives. The process use fresh beans, not having undergone a pre-treatment or defatting, having had their pulp and shell removed, in such a way as to obtain clean kernels. The grinding of said kernels in the presence of a solvent, the maceration of the ground kernels under conditions allowing the desired compounds to be extracted, the filtration of the maceration mixture, and the recovery of the extract containing said compounds from the filtrate.        9. US 2010 062138 is a method of selective adsorption of polyphenols on an adsorbent resin, using isopropanol as the solvent to make, cocoa extracts with 90% polyphenol content. Though the patent has quantified the polyphenol content, nothing is mentioned about the theobromine levels in the final product. It is also specific for unfermented cocoa beans having high levels of polyphenols and was blind about the fiber and proteins recovery, the method of application to fermented cocoa beans etc.        
The limitation of all the prior arts is that they all use specially processed unfermented or under fermented cocoa beans. The processes in the prior arts are not efficient enough to quantitatively isolate water soluble polyphenols from fermented cocoa beans containing relatively low amounts of polyphenols as compared to the unfermented cocoa beans. The fermented cocoa beans are rich in water insoluble procyanidins, which makes the prior art processes difficult. Pre-treatment of cocoa and its post-harvest operations for overcoming this difficulty constitute a major drawback, as it destroys the active healthy polyphenols of cocoa beans. This also makes the commercial extraction of polyphenols from common fermented cocoa beans difficult or even impossible. The prior arts isolate only one value added fraction, namely polyphenols or flavonoids of cocoa beans and leaves more than 90% (w/w) of cocoa beans as waste. None of the prior art provides a complete fractionation of the cocoa beans into value added products to utilize the whole components of cocoa as valuable products.
The present invention overcomes the limitations in the prior arts and is a ‘Green process’. The present invention also utilizes the waste after solvent extraction for making various value added products and also minimizes the use of organic solvents like hexane.
The prior art methodologies include multi solvent extraction methods designed for batch wise processes, essentially including initial defatting process of hexane extraction or supercritical fluid extraction followed by drying of cocoa residue and further polyphenol extraction with aqueous mixture of polar organic solvents. The disadvantage of the prior art is that these methods lack the integrity due to the use of class I solvents like hexane, which are not preferred in the manufacture of food ingredients and dietary supplements. Drying of hexane defatted cocoa beans is difficult in a commercial scale, as normal drying method using steam jacket, or electrical heating will damage the polyphenol content and cause colour change and aroma change due to the presence of various phytochemicals such as amines, fat, polyphenols etc. Yet another disadvantage associated with the prior arts is that the relative percentage of methylxanthins, especially theobromine and caffeine content of the isolated polyphenol rich extracts is not reported in many cases and hence it is not possible to develop methods for controlling their levels in the isolated polyphenol-rich extracts. All these prior arts consider only the subject compound leaving the majority of the other phytonutrients as waste.
The prior arts U.S. Pat. No. 6,627,232, U.S. Pat. No. 5,554,645, U.S. Pat. No. 4,390,698, U.S. Pat. No. 4,407,834, U.S. Pat. No. 4,755,391, U.S. Pat. No. 4,444,798, U.S. Pat. No. 4,755,391, U.S. Pat. No. 4,904,773, WO 2010/066015 A2, U.S. Pat. No. 1,073,441, U.S. Pat. No. 1,855,026, U.S. Pat. No. 1,925,326, U.S. Pat. No. 4,755,391, US 2003/0170199 A1 and US 2004/103334A1 were dealing with the extraction and purification of another group of phytonutrients called methylxanthines, especially theobromine present in cocoa beans. These prior arts dealing with the extraction and purification of methylxanthines, were mainly carried out in fermented cocoa beans and are very specific for the desired compound, leaving more than 90% of cocoa solids containing valuable dietary fiber and proteins as waste. Some prior arts have not accounted for the valuable antioxidant polyphenol load, while isolating the methylxanthines. The advantage of the present invention over these prior arts is that it provides a unique method for the quantitative extraction and purification of theobromine from cocoa beans, while allowing the complete fractionation of cocoa beans into other useful products such as cocoa polyphenols, dietary fiber, proteins etc. Further, the recovery of methylxanthines has been tremendously improved by using the technique of ultrasonication in commercial level.
Though many patents have been listed for the preparation and usefulness of other constituents of cocoa beans such as proteins, there is no prior art suggesting a green process for the complete fractionation of cocoa beans into commercially significant value-added products for food ingredient, functional food ingredient, nutraceutical, and/or cosmeceutical use. The present invention attempts to overcome the limitations of the prior arts and relates to a novel extraction method based on ultrasound technology employing enzymes, for the quantitative conversion of cocoa beans into various value-added ingredients. A state-of-the-art plant facility for executing the said process of ultrasound assisted continuous extraction and fractionation of extract into various value-added products in commercial scale was also provided. PLC—(Programmable Logic Controller) based ultrasonication was developed for automation of the process at controlled temperature. The present invention deals with specific conditions for such extractions and use of enzymes in such extractions for enhancing the recovery of active ingredients such as polyphenols from cocoa beans.
An advantage of the present invention is that it relates to a novel extraction method based on ultrasound technology employing enzymes, for the quantitative conversion of cocoa beans into various value-added functional ingredients and a total state-of-the-art plant facility for executing the process in commercial scale, and continuous manner with PLC-based ultrasonication at critical steps such as initial cocoa beans extraction and soluble dietary fiber extraction. Ultrasonication at critical steps helps for efficient extraction of phytochemicals such as polyphenols and methylxanthines, with more than 95% recovery. The invention also has the advantage in that the process can be applied to both fermented, unfermented or under fermented cocoa beans for quantitative isolation of available phytonutrients. Ultrasonication also helps the direct extraction of cocoa beans without prior defatting processes like hexane extraction or mechanical expulsion or supercritical extraction or the like, which usually form an important step in all the prior arts. The use of various enzymes in combination with ultrasonication helps for the efficient extraction and preparation of soluble dietary fiber in high yield and purity.
Another advantage of the present invention over the prior arts was that it has used various enzymes in combination with ultrasonication for the efficient extraction and preparation of soluble dietary fiber from cocoa nibs, in high yield and purity.
Yet another advantage of the present invention over the prior arts is that the present process can avoid use of multiple solvents and total extraction of phytonutrients can be achieved on non-defatted cocoa beans.
A further advantage of the present invention over the prior arts is that better purity and extraction yield and fast cycle time are achieved.
Still another advantage of the present invention over the prior arts is that the present process can be automated for selective application of ultrasound at various stages of operations, resulting in the continuous production of various cocoa beans involving minimum labour and cost.
Another advantage of the present invention over the prior arts is that various products are produced simultaneously and continuously and the total design of a plant facility for such a production of various value added products is achieved.
A further advantage of the present invention over the prior arts is that the present process uses simple water based ion exchange and adsorption chromatography for the purification and recovery of phytonutrients like polyphenols and methylxanthins in single operations.
Yet another advantage of the present process is to make various grades of methylxanthins and its unique compositions with epicatechin by selectively eliminating higher oligomeric procyanidins, using a liquid-liquid extraction process. Epicatechin is regarded as one of the efficient bioavailable antioxidant polyphenol belonging to flavan-3-ol widely distributed in cocoa and many other plants.
Another advantage of the present invention over the prior arts is that the residue of cocoa nibs left after cocoa flavor and polyphenol extraction is further processed to dietary fiber rich in proteins with unique flavor suitable for using in cocoa or chocolate based food products.
Still another advantage of the present invention over the prior arts is that the present process can produce high yield and purity, not less than 70% of cocoa soluble dietary fiber from the residue of cocoa nibs left after cocoa flavor or phytonutrients extraction. High soluble fiber content helps to reduce the dosage and easy incorporation into various food stuffs at physiologically significant dosages per serving. Moreover, higher the purity of the fiber, higher will be its neutral organoleptic character, such as taste and flavor/aroma. Consumption of physiologically significant amounts of soluble fiber helps to reduce cholesterol levels, blood sugar levels, for satiety, to reduce hunger etc.
Yet another advantage of the present invention over the prior arts is that cocoa nibs derived soluble dietary fiber is employed for the encapsulation of cocoa polyphenols to enhance the stability, solubility and bioavailability. Being a soluble dietary fiber derived from cocoa, it can be used as a very safe fiber supplement in all cocoa or chocolate containing food items. It can be used as a replacement for many other excipients/additives like starch, hydroxypropylmethylcellulose, and other thickeners, hydrocolloids and binders like guar gum, gum acacia etc. Being a byproduct in cocoa polyphenol or cocoa flavor production, it becomes cost effective. It also provides a unique way of preparation of antioxidant dietary fiber or dietary fiber possessing good antioxidant properties for dietary supplement applications.
A further advantage of the present invention is that it provides a state-of-the-art plant facility where by cost effective commercial scale continuous process of extraction of cocoa beans can be performed by employing PLC-controlled automatic ultrasonication for fast and efficient extraction.
Yet another advantage of the present invention is that it provides a methodology for applying ultrasound waves to a continuous extractor and batch extractor for enhancing the efficiency of botanical extractions.
Another advantage of the present invention is that it efficiently uses ultrasonication for efficient extraction of phytochemicals such as polyphenols and methyxanthines from lipid rich plant seeds such as cocoa beans.